Polynucleotide constructs, pharmaceutical compositions and methods for targeted downregulations of angiogenesis and anticancer therapy

ABSTRACT

A novel nucleic acid construct for down-regulating angiogenesis in a tissue of a subject is provided. The nucleic acid construct includes: (a) a first polynucleotide region encoding a chimeric polypeptide including a ligand binding domain fused to an effector domain of an apoptosis signaling molecule; and (b) a second polynucleotide region encoding a cis acting regulatory element being for directing expression of the chimeric polypeptide in a specific tissue or cell; wherein the ligand binding domain is selected such that it is capable of binding a ligand present in the specific tissue or cell, whereas binding of the ligand to the ligand binding domain activates the effector domain of the apoptosis signaling molecule. Also provided are methods of utilizing this nucleic acid construct for treating diseases characterized by excessive or aberrant neo-vascularization or cell growth.

RELATED APPLICATION/S

This application is a Divisional of U.S. patent application Ser. No.13/163,767, filed on Jun. 20, 2011, which is a Divisional of U.S. patentapplication Ser. No. 12/222,439 filed on Aug. 8, 2008, now U.S. Pat. No.7,989,427, which is a Divisional of U.S. patent application Ser. No.10/490,746 filed on Apr. 12, 2004, now U.S. Pat. No. 7,585,666, which isa National Phase of PCT Patent Application No. PCT/IL02/00339 havingInternational filing date of May 1, 2002, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 60/330,118 filed onOct. 19, 2001. The contents of the above applications are allincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 3182_(—)0360004_SequenceListing_ascii; Size: 3,365bytes; and Date of Creation: Mar. 5, 2013), filed herewith, isincorporated herein by reference in its entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a nucleic acid constructs,pharmaceutical compositions and methods which can be used todownregulate angiogenesis in specific tissue regions of a subject. Moreparticularly, the present invention relates to nucleic acid constructs,which can be used to activate apoptosis in specific cell subsets, thus,enabling treatment of diseases characterized by excessive or aberrantneovascularization or cell growth.

Angiogenesis is the growth of new blood vessels, a process that dependsmainly on locomotion, proliferation, and tube formation by capillaryendothelial cells. During angiogenesis, endothelial cells emerge fromtheir quiescent state and proliferate rapidly. Although the molecularmechanisms responsible for transition of a cell to angiogenic phenotypeare not known, the sequence of events leading to the formation of newvessels has been well documented [Hanahan, D., Science 277, 48-50,(1997)]. The vascular growth entails either endothelial sprouting[Risau, W., Nature 386, 671-674, (1997)] or intussusceptions [Patan, S.,et al; Microvasc. Res. 51, 260-272, (1996)]. In the first pathway, thefollowing sequence of events may occur: (a) dissolution of the basementof the vessel, usually a post capillary venule, and the interstitialmatrix; (b) migration of endothelial cells toward the stimulus; (c)proliferation of endothelial cells trailing behind the leadingendothelial cell (s); (d) formation of lumen (canalization) in theendothelial array/sprout; (e) formation of branches and loops byconfluencial anastomoses of sprouts to permit blood flow; (f) investmentof the vessel with pericytes (i.e., periendothelial cells and smoothmuscle cells); and (g) formation of basement membrane around theimmature vessel. New vessels can also be formed via the second pathway:insertion of interstitial tissue columns into the lumen of preexistingvessels. The subsequent growth of these columns and their stabilizationresult in partitioning of the vessel lumen and remodeling of the localvascular network.

A variety of angiogenic factors govern the angiogenic process. It isunderstood that during pathology, the fine balance betweenpro-angiogenic factors and anti-angiogenic factors is disrupted, therebyeliciting nonself-limiting endothelial and periendothelialcell-proliferation. Until recently, the angiogenesis that occurs indiseases of ocular neovascularization, arthritis, skin diseases, andtumors, had been difficult to suppress therapeutically.

Therefore, the fundamental goal of all anti-angiogenic therapy is toreturn foci of proliferating microvessels to their normal resting state,and to prevent their regrowth [Cancer: Principles & Practice ofOncology, Fifth Edition, edited by Vincent T. DeVita, Jr., SamuelHellman, Steven A. Rosenberg. Lippincott-Raven Publishers, Philadelphia.(1997)].

Anti-angiogenic therapy is a robust clinical approach, as it can delaythe progression of tumor growth (e.g., retinopathies, benign andmalignant angiogenic tumors).

In general, every disease caused by uncontrolled growth of capillaryblood vessels such as diabetic retinopathy, psoriasis, arthritis,hemangiomas, tumor growth and metastasis is a target for anti-angiogenictherapy.

For example, the progressive growth of solid tumors beyond clinicallyoccult sizes (e.g., a few mm³) requires the continuous formation of newblood vessels, a process known as tumor angiogenesis. Tumor growth andmetastasis are angiogenesis-dependent. A tumor must continuouslystimulate the growth of new capillary blood vessels to deliver nutrientsand oxygen for the tumor itself to grow. Therefore, either prevention oftumor angiogenesis or selective destruction of tumor's existing bloodvessels (vascular targeting therapy) underlies anti-angiogenic tumortherapy.

Recently, a plethora of anti-angiogenic agents has been developed forthe treatment of malignant diseases, some of which are already underclinical trials (for review see Herbst et al. (2002) Semin. Oncol.29:66-77).

The most studied target for tumor anti-angiogenic treatment is thedominant process regulating angiogenesis in human i.e., the interactionof vascular endothelial growth factor (VEGF) with its receptor (VEGFR).Agents which regulate VEGFR pro-angiogenic action include (i) antibodiesdirected at the VEGF protein itself or to the receptor (e.g., rhuMAbVEGF); (ii) small molecule compounds directed to the VEGFR tyrosinekinase (e.g., ZD6474 and SU5416); (iii) VEGFR targeted ribozymes.

Other novel angiogenesis inhibitors include 2-Methoxyestradiol (2-ME2) anatural metabolite of estradiol that possesses unique anti-tumor andanti-angiogenic properties and angiostatin and endostatin-proteolyticcleavage fragments of plasminogen and collagen XVIII, respectively.

Though promising in pre-clinical models, to date systemic administrationof all anti-angiogenic agents tested in clinical trials, have shownlimited rate of success and considerable toxicities includingthrombocytopenia, leukopenia and hemoptysis. These results suggest thatthere may be limits to the use of current tumor anti-angiogenic agentsas therapy for advanced malignancies. O'Reilly et al. have shown thatthe latency between the initiation of anti-angiogenic therapy andantitumor effect may result in initial tumor progression before responseto therapy [O'Reilly S et al. (1998) Proc Am Soc Clin Oncol 17:217a].Furthermore, recent studies suggest that the regulation of angiogenesismay differ among capillary beds, suggesting that anti-angiogenic therapymay need to be optimized on an organ/tissue-specific basis [Arap et al.(1998) Science 279:377-380].

Interestingly, poor results have also been obtained when anti-angiogenictherapy (e.g., heparin, heparin-peptide treatment) directed at smoothmuscle cell to proliferation has been practiced on myocardial ischemiain patients with coronary artery disease [Liu et al., Circulation, 79:1374-1387 (1989); Goldman et al., Atherosclerosis, 65: 215-225 (1987);Wolinsky et al., JACC, 15 (2): 475-481 (1990)]. Various limitationsassociated with the use of such agents for the treatment ofcardiovascular diseases included: (i) systemic toxicity creatingintolerable level of risk for patients with cardiovascular diseases;(ii) interference with vascular wound healing following surgery; (iii)possible damage to surrounding endothelium and/or other medial smoothmuscle cells.

In-light of these and the inherent obstacles associated with systemicadministration of anti-angiogenic factors (i.e., manufacturinglimitations based on in-vitro instability and high doses required; andpeak kinetics of bolus administration attributing to sub-optimaleffects) limit the effective use of angiogenic factors in treatingneo-vascularization associated diseases.

With the identification of new genes that regulate the angiogenicprocess, somatic gene therapy has been attempted to overcome theselimitations. Although, great efforts have been directed towardsdeveloping methods for gene therapy of cancer, cardiovascular andperipheral vascular diseases, there is still major obstacles toeffective and specific gene delivery [for review see, Feldman A L.(2000) Cancer 89(6):1181-94] In general, the main limiting factor ofgene therapy with a gene of interest, using a recombinant viral vectoras a shuttle is the ability to specifically direct the gene of interestto the target tissue.

Attempts to overcome these limitations included the use oftissue-specific promoters conjugated to cytotoxic genes. For example,endothelial cell targeting of a cytotoxic gene, expressed under thecontrol endothelial-specific promoters has been described by Jagger etal who used the KDR or E-selectin promoter to express TNFα specificallyin endothelial cells [Jaggar R T. Et al. Hum Gene Ther (1997)8(18):2239-47]. Ozaki et al used the von-Willebrand factor (vWF)promoter to deliver herpes simplex virus thymidine kinase (HSV-tk) toHUVEC [Hum Gene Ther (1996) 7(13):1483-90]. However, these promotersshowed only weak activity and did not allow for high levels ofexpression.

An alternate approach presented by Kong and Crystal included a tumorspecific expression of anti-angiogenic factors. To date, however, thetoxicity of recombinant forms of endogenous anti-angiogenic agents hasnot been demonstrated although some synthetic anti-angiogenic agentshave been associated with toxicity in preclinical models [Kong andCrystal (1998) J. Natl. Cancer Inst. 90:273-76].

Angiostatin has also been used as a possible anti-angiogenic agent(Falkman et al, Cell 1997 Jan. 24; 88(2):277-85), however due to theredundancy of factors involved in regulation of angiogenesis in tumors,it is highly unlikely that angiostatin therapy alone would be effective.

There is thus a widely recognized need for, and it would be highlyadvantageous to have a novel approach for efficiently down-regulatingangiogenesis in specific tissue regions of a subject while being devoidof the toxic side effects characterizing prior art anti-angiogenesisapproaches.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anucleic acid construct comprising: (a) a first polynucleotide regionencoding a chimeric polypeptide including a ligand binding domain fusedto an effector domain of an apoptosis signaling molecule; and (b) asecond polynucleotide region encoding a cis acting regulatory elementbeing for directing expression of the chimeric polypeptide in a specifictissue or cell; wherein the ligand binding domain is selected such thatit is capable of binding a ligand present in the specific tissue orcell, whereas binding of the ligand to the ligand binding domainactivates the effector domain of the apoptosis signaling molecule.

According to further features in preferred embodiments of the inventiondescribed below, there is provided a mammalian cell transformed with thenucleic acid construct described above.

According to another aspect of the present invention there is provided amethod of down-regulating angiogenesis in a tissue of a subject, themethod comprising administering to the subject a nucleic acid constructdesigned and configured for generating apoptosis in a sub-population ofangiogenic cells, the nucleic acid construct including: (a) a firstpolynucleotide region encoding a chimeric polypeptide including a ligandbinding domain fused to an effector domain of an apoptosis signalingmolecule; and (b) a second polynucleotide region encoding a cis actingregulatory element being for directing expression of the chimericpolypeptide in the sub-population of angiogenic cells; wherein theligand binding domain is selected such that it is capable of binding aligand present in, or provided to, the sub-population of angiogeniccells, whereas binding of the ligand to the ligand binding domainactivates the effector domain of the apoptosis signaling molecule,thereby down-regulating angiogenesis in the tissue.

According to further features in preferred embodiments of the inventiondescribed below, the method further comprising administering the ligandto the subject in a manner suitable for providing the ligand to thesub-population of angiogenic cells.

According to yet another aspect of the present invention there isprovided method of down-regulating angiogenesis in a tissue of asubject, the method comprising: (a) administering to the subject anucleic acid construct designed and configured for generating apoptosisin a sub-population of angiogenic cells, the nucleic acid constructincluding: (i) a first polynucleotide region encoding a chimericpolypeptide including a ligand binding domain fused to an effectordomain of an apoptosis signaling molecule, wherein the effector domainis selected such that it is activated following binding of a ligand tothe ligand binding domain; and (ii) a second polynucleotide regionencoding a cis acting regulatory element being for directing expressionof the chimeric polypeptide in the sub-population of angiogenic cells;and (b) administering to the subject the ligand, thereby down-regulatingangiogenesis in the tissue.

According to still further features in the described preferredembodiments the administering the ligand to the subject is effected by amethod selected from the group consisting of: (i) systemic in-vivoadministration; (ii) ex-vivo administration to cells removed from a bodyof the subject, the cells subsequently reintroduced into the body of thesubject; and (iii) local in-vivo administration.

According to still further features in the described preferredembodiments the cis-acting regulatory element is an endothelialcell-specific or periendothelial cell-specific promoter selected fromthe group consisting of the PPE-1 promoter, the PPE-1-3x promoter, theTIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the vonWillerband promoter, the KDR/flk-1 promoter, The FLT-1 promoter, theEgr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1promoter, the CArG box element and aortic carboxypeptidase-like protein(ACLP) promoter.

According to still further features in the described preferredembodiments the ligand binding domain is a ligand-binding domain of acell-surface receptor.

According to still further features in the described preferredembodiments the cell-surface receptor is selected from the groupconsisting of a receptor tyrosine kinase, a receptor serine kinase, areceptor threonine kinase, a cell adhesion molecule and a phosphatasereceptor.

According to still further features in the described preferredembodiments the apoptosis signaling molecule is selected from the groupconsisting of Fas and TNFR.

According to still another aspect of the present invention there isprovided a pharmaceutical composition for down regulating angiogenesisin a tissue of a subject, the pharmaceutical composition comprising asan active ingredient a nucleic acid construct designed and configuredfor generating apoptosis in a subpopulation of angiogenic cells and apharmaceutical acceptable carrier, the nucleic acid construct including:(a) a first polynucleotide region encoding a chimeric polypeptideincluding a ligand binding domain fused to an effector domain of anapoptosis signaling molecule; and (b) a second polynucleotide regionencoding a cis acting regulatory element being for directing expressionof the chimeric polypeptide in the subpopulation of angiogenic cells;wherein the ligand binding domain is selected such that it is capable ofbinding a ligand present in the specific tissue or cell, whereas bindingof the ligand to the ligand binding domain activates the effector domainof the apoptosis signaling molecule.

According to an additional aspect of the present invention there isprovided a method of treating a disease or condition associated withexcessive neo-vascularization, the method comprising administering atherapeutically effective amount of a nucleic acid construct designedand configured for generating apoptosis in a sub-population ofangiogenic cells, the nucleic acid construct including: (i) a firstpolynucleotide region encoding a chimeric polypeptide including a ligandto binding domain fused to an effector domain of an apoptosis signalingmolecule; and (ii) a second polynucleotide region encoding a cis actingregulatory element being for directing expression of the chimericpolypeptide in the sub-population of angiogenic cells; wherein theligand binding domain is selected such that it is capable of binding aligand present in, or provided to, the sub-population of angiogeniccells, whereas binding of the ligand to the ligand binding domainactivates the effector domain of the apoptosis signaling molecule,thereby down-regulating angiogenesis in the tissue and treating thedisease or condition associated with excessive neo-vascularization.

According to yet an additional aspect of the present invention there isprovided a method of treating a tumor in a subject, the methodcomprising administering a therapeutically effective amount of a nucleicacid construct designed and configured for generating apoptosis in cellsof the tumor, the nucleic acid construct including: (i) a firstpolynucleotide region encoding a chimeric polypeptide including a ligandbinding domain fused to an effector domain of an apoptosis signalingmolecule; and (ii) a second polynucleotide region encoding a cis actingregulatory element being for directing expression of the chimericpolypeptide in the cells of the tumor; wherein the ligand binding domainis selected such that it is capable of binding a ligand present in, orprovided to, the cells of the tumor, whereas binding of the ligand tothe ligand binding domain activates the effector domain of the apoptosissignaling molecule to thereby direct apoptosis in the cells of thetumor.

According to still further features in the described preferredembodiments the method further comprising administering the ligand tothe subject in a manner suitable for providing the ligand to the cellsof the tumor.

According to still further features in the described preferredembodiments the cis acting regulatory element is selected from the groupconsisting of the gastrin-releasing peptide (GRP) promoter, the hTERTpromoter, the Hexokinase type II promoter and the L-plastin promoter.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a novel approach forefficiently downregulating angiogenesis and tumor cell proliferation ina specific and targeted manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the Drawings:

FIGS. 1 a-b are schematic illustrations of Fas chimera gene constructedfrom the extracellular region of TNFR1 and the trans-membrane andintracellular regions of Fas and cloned into pcDNA3 plasmid (a) or intoadenoviral vectors (b).

FIGS. 2 a-b illustrate apoptotic activity of the pro-apoptotic genes,Fas chimera and TNFR1. FIG. 2 a—illustrates Bovine Aortic EndothelialCells (BAEC) transfected with either pcDNA-3-TNFR1 (lower panel) orcontrol empty vector (upper panel) and an expression plasmid encodingGFP. FIG. 2 b—illustrates 293 Cells transfected with eitherpcDNA-3-Fas-c (lower panel) or control empty vector (upper panel) and anexpression plasmid encoding GFP. Transfected cells were visualized usingfluorescence microscopy and apoptotic activity was morphologicallydetermined.

FIGS. 3 a-f are electron microscopy images of BAEC cells transfectedwith pro-apoptotic genes. 24 hours post transfection, BAEC cells werefixed in 2.5% glutaraldehyde and processed. Presented are cells insuccessive stages of the apoptotic process.

FIG. 4 are histograms quantifying apoptotic activity of the indicatedpro-apoptotic genes in transfected BAEC and 293 cells.

FIG. 5 a represents a PCR analysis of AdPPE-Fas-c. Lanes 1-2—PCRproducts obtained using primers encompassing the PPE-1 promoter andFas-c gene. Lanes 3-4—PCR products obtained using Fas-c primers. Lanes5-6—PCR products obtained in the absence of template DNA.

FIG. 5 b is a western blot analysis of AdPPE-Fas-c transfected BAECcells. Protein samples were resolved by SDS-PAGE, transferred tonitrocellulose membrane and probed with a polyclonal antibody directedagainst the extracellular portion of TNFR1. Lane 1-2—pcDNA3-Fas-c BAECtransfected cells (positive control). Lane 3-4—BAEC cells transfectedwith the indicated MOI of AdPPE-Fas-c viruses. Lane 5—non-transfectedcells. Lane 6-7—BAEC cells transfected with the indicated MOI ofAdPPE-Luc.

FIGS. 6 a-d are photomicrographs illustrating the effect of Fas-chimeraover-expression on apoptosis of endothelial cells. BAEC cells wereinfected with: Ad-PPE-1-3x-Fas-chimera (FIG. 6 a);Ad-PPE-1-3x-luciferase (FIG. 6 b); Ad-PPE-1-3x-Fas-chimera andAd-PPE1-3x-GFP (FIG. 6 c); Ad-PPE-1-3x-luciferase and Ad-PPE-1-3x-GFP;each at MOI 1000 (FIG. 6 d). Photomicrographs were taken 72 h postinfection at ×10 magnification.

FIG. 7 is a histogram illustrating apoptotic specific effect ofAd-PPE-1-3x-Fas-chimera on endothelial cells. Viability of endothelial(BAEC, HUVEC) and non-endothelial (Normal skin fibroblasts-NSF) cellswas quantified by crystal violet staining 72 h post infection witheither Ad-PPE-1-3x-Fas-chimera or control (luciferase) virus.

FIG. 8 shows a dose response effect of TNFα administration onFas-chimera mediated apoptosis. BAEC were infected withAd-PPE-1-3x-Fas-c. 48 h post infection TNF was added to the growthmedium (at the indicated dose). Viability was determined by the crystalviolet assay 24 h thereafter.

FIGS. 9 a-e are photomicrographs illustrating an endothelialcell-specific apoptosis mediated by the cooperative action of TNFαligand and Fas-c receptor. The indicated cells were incubated in thepresence or absence of TNFα (10 ng/ml) 48 h following infection withAd-PPE-1-3x-Fas-c; crystal violet staining was effected 72 h postinfection.

FIG. 10 a is a dose response curve illustrating the TNFα-dependentapoptotic effect of Ad-CMV-Fas-c on endothelial cells. Viability of BAECcells infected with the indicated MOI of Ad-CMV-Fas-chimera wasdetermined following incubation with TNFα.

FIGS. 10 b-d illustrate the apoptotic effect of TNFα ligand andAd-CMV-Fas-chimera on the non-endothelial cells NSF. FIG. 10 b—NSFinfected with a control virus. FIG. 10 c—NSF infected withAd-CMV-Fas-chimera. FIG. 10 d—NSF infected with Ad-CMV-Fas-chimera andincubated with TNF (10 ng/ml).

FIGS. 11 a-c illustrate the In-vivo anti-tumoral effect ofAd-PPE-1-3x-Fas-c. Mice inoculated with B16 melanoma cells were injectedintravenously with Ad-PPE-1-3x-Fas-c, Ad-CMV-Fas-chimera, control virusor saline when tumor was palpable. FIG. 11 a—tumor areas, measuredduring treatment period. FIG. 11 b—tumor weights at end of treatmentperiod. FIG. 11 c—an image representing the state of the tumor in theAd-PPE-1-3x-Fas-c treated mouse and the control mouse.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of nucleic acid constructs and methods, whichcan be used to treat diseases characterized by excessive or aberrantneovascularization or cell growth. Specifically, the present inventioncan be used to specifically target and kill cells involved inangiogenesis, thus enabling down-regulation of angiogenesis andanti-tumor therapy.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

One of the most fundamental goals of anti-angiogenic therapy is toreturn foci of proliferating microvessels to their normal resting state,and to prevent their re-growth.

To date, anti-angiogenic therapy approaches, which employ systemicadministration of anti-angiogenic agents have been employed with limitedsuccess mostly due to the toxic side effects which lead to the formationof thrombocytopenia, leukopenia and/or hemoptysis in the treatedindividual. The toxic side effects associated with prior art approachesis a result of non-specific expression of the anti-angiogenic agentsemployed and exposure of healthy tissue to these agents or theredundancy and thus non-effectiveness of the anti-angiogenic agentsused.

While reducing the present invention to practice, the present inventorshave uncovered that a combination of tissue-specific expression andspecific activation of a pro-apoptotic agent enables selective apoptosisof cells involved in angiogenesis without exposing non-targeted tissueor cells to these agents, thus, avoiding the toxic side effects andredundancy characterizing prior art treatment approaches.

Thus, according to one aspect of the present invention there is provideda method of down-regulating angiogenesis in a tissue of a subject. Asused herein, the phrase “down-regulating angiogenesis” refers to eitherslowing down or stopping the angiogenic process, which lead to formationof new blood vessels.

The method according to this aspect of the present invention is effectedby administering to the subject a nucleic acid construct designed andconfigured for generating apoptosis in a sub-population of angiogeniccells. As used herein, the phrase “angiogenic cells” refers to anycells, which participate or contribute to the process of angiogenesis.Thus, angiogenic cells include but are not limited to, endothelialcells, smooth muscle cells.

In order to direct specific expression of an apoptotic agent in asubpopulation of angiogenic cells, the nucleic acid construct of thepresent invention includes a first polynucleotide region encoding achimeric polypeptide including a ligand binding domain which can be, forexample, a cell-surface receptor domain of a receptor tyrosine kinase, areceptor serine kinase, a receptor threonine kinase, a cell adhesionmolecule or a phosphatase receptor fused to an effector domain of anapoptosis signaling molecule such as, for example, Fas, TNFR, and TRAIL.

Such a chimeric polypeptide can include any ligand binding domain fusedto any apoptosis signaling domain as long as activation of the ligandbinding domain, i.e., via ligand binding, triggers apoptosis signalingvia the effector domain of the apoptosis signaling molecule.

Selection of the ligand binding domain and the apoptosis signalingdomain fused thereto is affected according to the type of angiogeniccell targeted for apoptosis. For example, when targeting specific subsetof endothelial cells (e.g., proliferating endothelial cells, orendothelial cells exhibiting a tumorous phenotype), the chimericpolypeptide includes a ligand binding domain capable of binding a ligandnaturally present in the environment of such endothelial cells andpreferably not present in endothelial cells of other non-targetedtissues (e.g., TNF, VEGF). Such a ligand can be secreted by endothelialcells (autocrine), secreted by neighboring tumor cells (paracrine) orspecifically targeted to these endothelial cells.

Examples of suitable chimeric polypeptides are provided in Examples 2 ofthe Examples section which follows. Preferably, the chimeric polypeptideis the Fas-c chimera which is described in detail in Examples 2-4 of theExamples section which follows.

The use of such a chimeric polypeptide is particularly advantageous,since, as shown in the Examples section hereinunder, it enablesefficient and robust activation of apoptosis in a specific subset ofangiogenic cells while avoiding activation in other subset of cells,which are not targeted for apoptosis.

To further enhance cell specificity of apoptosis, the nucleic acidconstruct of the present invention further includes a secondpolynucleotide region, which encodes a cis acting regulatory element(e.g., promoter and/or enhancer) capable of directing expression of thechimeric polypeptide in the sub-population of angiogenic cells.

Examples of suitable promoters/enhancers which can be utilized by thenucleic acid construct of the present invention include theendothelial-specific promoters: preproendothelin-1, PPE-1 promoter(Harats D, J Clin Invest. 1995 March; 95(3):1335-44)., the PPE-1-3xpromoter [PCT/IL01/01059; Varda-Bloom N, Gene Ther 2001 June;8(11):819-27], the TIE-1 (S79347, S79346) and the TIE-2 (U53603)promoters [Sato T N, Proc Natl Acad Sci USA 1993 Oct. 15;90(20):9355-8], the Endoglin promoter [Y11653; Rius C, Blood 1998 Dec.15; 92(12):4677-90], the von Willerbrand factor [AF152417; Collins C JProc Natl Acad Sci USA 1987 July; 84(13):4393-7], the KDR/flk-1 promoter[X89777, X89776; Ronicke V, Circ Res 1996 August; 79(2):277-85], TheFLT-1 promoter [D64016 AJ224863; Morishita K: J Biol Chem 1995 Nov. 17;270(46):27948-53], the Egr-1 promoter [AJ245926; Sukhatme V P, OncogeneRes 1987 September-October; 1(4):343-55], the E-selectin promoter[Y12462; Collins T J Biol Chem 1991 Feb. 5; 266(4):2466-73], Theendothelial adhesion molecules promoters: ICAM-1 [X84737; Horley K JEMBO J 1989 October; 8(10):2889-96], VCAM-1 [M92431; lademarco M F, JBiol Chem 1992 Aug. 15; 267(23):16323-9], PECAM-1 [AJ313330×96849; CD31,Newman P J, Science 1990 Mar. 9; 247(4947):1219-22], the vascularsmooth-muscle-specific elements: CArG box X53154 and aorticcarboxypeptidase-like protein (ACLP) promoter [AF332596; Layne M D, CircRes. 2002; 90: 728-736] and Aortic Preferentially Expressed Gene-1[Yen-Hsu Chen J. Biol. Chem., Vol. 276, Issue 50, 47658-47663, Dec. 14,2001].

Preferably, the promoter utilized by the construct of the presentinvention is functional in proliferating angiogenic cells, or angiogeniccells of a particular phenotype (e.g., tumorous). A promoter highlysuitable for use with the present invention is the PPE-1-3x promoterwhich is further described in the Examples section which follows. Avector construct most suitable for use with the present invention is thewidely used adenoviral vector and its derivatives.

It was discovered that a novel configuration of the PPE-1 enhancersequence of the present invention endows promoter sequences with anunexpected and highly specific activity in endothelial cellsparticipating in angiogenesis. Thus, according to one aspect of thepresent invention there is provided an isolated polynucleotidefunctional as an endothelial cell specific promoter in a mammal such asa human being. The isolated polynucleotide includes an enhancer elementincluding one or more copies of the sequence set forth in SEQ ID NO:6and preferably one or more copies of the sequence set forth in SEQ IDNO: 8, which as is illustrated in the Examples section which follows,plays an important role in regulating expression in endothelial cellsparticipating in angiogenesis. One specific and novel sequenceconfiguration of an enhancer element utilizable by the present inventionis illustrated in SEQ ID NO:7.

For purposes of this specification and the accompanying claims, the term“enhancer” refers to any polynucleotide sequence which increases thetranscriptional efficiency of a promoter.

According to some embodiments of the invention, the isolatedpolynucleotide includes contiguous copies of SEQ ID NOs:6 and/or 8. Suchsequences are preferably positioned in a head-to-tail orientation,although, the enhancer element of the present invention can also includeone or more copies of a specific portion of the sequence of SEQ ID NO:6or 8, in an inverted orientation, e.g., by using sequences complementaryto SEQ ID NO:6 or 8 in construction of the enhancer element.

Thus, it is postulated that a minimal configuration of an enhancerelement according to the present invention is an isolated polynucleotideas set forth in SEQ ID NO: 8. This enhancer is anticipated to functionwith a wide variety of promoters, including but not limited toendothelial specific promoters (e.g. PPE-1; SEQ ID NO.: 1) andconstitutive promoters, for example viral promoters such as thosederived from CMV and SV-40. This enhancer should be capable of impartingendothelial specificity to a wide variety of promoters. The enhancerelement may be augmented, for example by addition of one or more copiesof the sequence set forth in SEQ ID NO:6. These additional sequences maybe added contiguously or non-contiguously to the sequence of SEQ ID NO.:8.

The present invention further includes a method of expressing a nucleicacid sequence of interest in endothelial cells employing a constructwhich relies upon an enhancer element including at least one copy of thesequence set forth in SEQ ID NO: 8 and a promoter to direct high levelexpression of the sequence of interest specifically to endothelialcells.

The promoter sequences generated according to the teachings of thepresent invention are particularly useful in regulating angiogenesis ina tissue. As illustrated in the Examples section which follows, themodified 3X (SEQ. ID. NO:7) containing promoter sequence of the presentinvention and the unmodified PPE-1 promoter are both expressed inmetastatic foci of the LLC model. Thus, use of a construct including the3X element in a gene therapy context can be expected to maximizedelivery to tumors while minimizing toxic effects on surrounding normaltissue.

The nucleic acid construct of the present invention can further includeadditional polynucleotide sequences such as for example, sequencesencoding selection markers or reporter polypeptides, sequences encodingorigin of replication in bacteria, sequences that allow for translationof several proteins from a single mRNA (IRES), sequences for genomicintegration of the promoter-chimeric polypeptide encoding region and/orsequences generally included in mammalian expression vector such aspcDNA3, pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto,pCMV/myc/cyto, pCR3.1, which are available from Invitrogen, pCI which isavailable from Promega, pBK-RSV and pBK-CMV which are available fromStratagene, pTRES which is available from Clontech, and theirderivatives.

Preferably, the nucleic acid construct of the present invention isadministered to the subject via, for example, systemic administrationroutes or via oral, rectal, transmucosal (especially transnasal),intestinal or parenteral administration routes. Systemic administrationincludes intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, intraocular injections or intra-tumoral.

Preferably, the subject is a mammal, more preferably, a human being,most preferably, a human being suffering from diseases characterized byexcessive or abnormal neovascularization such as that characterizingtumor growth, proliferating diabetic retinopathy, arthritis and thelike.

The nucleic acid constructs of the present invention can be administeredto the subject per se or as part (active ingredient) of a pharmaceuticalcomposition.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver naked or carrier provided polynucleotidesinto a wide variety of cell types (see, for example, Luft (1998) J MolMed 76(2): 75-6; Kronenwett et al. (1998) Blood 91(3): 852-62; Rajur etal. (1997) Bioconjug Chem 8(6): 935-40; Lavigne et al. (1997) BiochemBiophys Res Commun 237(3): 566-71 and Aoki et al. (1997) Biochem BiophysRes Commun 231(3): 540-5).

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients or agents described herein withother chemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered nucleic acid construct. An adjuvantis included under these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.In the context of the present invention, administration directly intotumor tissue is a relevant example of local administration.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredient of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (e.g. antisense oligonucleotide) effective toprevent, alleviate or ameliorate symptoms of a disorder (e.g.,progressive loss of bone mass) or prolong the survival of the subjectbeing treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin an animal model, such as the murine Src deficient model ofosteopetrosis (Boyce et al. (1992) J. Clin. Invest. 90, 1622-1627; Loweet al. (1993) Proc. Natl. Acad. Sci. USA 90, 4485-4489; Soriano et al.(1991) Cell 64, 693-702), to achieve a desired concentration or titer.Such information can be used to more accurately determine useful dosesin humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to levels of theactive ingredient are sufficient to retard tumor progression (minimaleffective concentration, MEC). The MEC will vary for each preparation,but can be estimated from in vitro data. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. Detection assays can be used to determine plasmaconcentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks ordiminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

The pharmaceutical compositions of the present invention may furtherinclude any additional ingredients which may improve the uptake of thenucleic acid construct by the cells, expression of the chimericpolypeptide encoded by the nucleic acid construct in the cells, or theactivity of the expressed chimeric polypeptide.

For example, the uptake of adenoviral vectors into EC cells can beenhanced by treating the vectors with engineered antibodies or smallpeptides. Such “adenobody” treatment, was shown effective in directingadenovirus constructs to EGF receptors on cells (Watkins et al 1997,Gene Therapy 4:1004-1012). In addition, Nicklin et al have shown that Asmall peptide, isolated via phage display, increased specificity andefficiency of vectors in endothelial cells and decreased the expressionin liver cells in culture (Nicklin et al 2000, Circulation 102:231-237).In a recent study, an FGF retargeted adenoviral vector reduced thetoxicity of tk in mice (Printz et al 2000, Human Gene Therapy11:191-204).

It will be appreciated that although targeting of cells exposed to theligand is preferred, the present invention also envisages expression ofthe nucleic acid construct of the present invention in cells which arenot exposed to, or naturally affected by the ligand. In such cases, themethod of the present invention includes the step of administering sucha ligand to the cells transformed. Such administration can be effectedby using any of the above described administration methods. Preferably,the ligand is administrated in a cell targeted manner, using for exampleantibody conjugated targeting, such that activation of apoptosissignaling is highly specific. This approach of apoptosis activation isdescribed in more detail in the Examples section which follows.

Thus, the present invention provides nucleic acid constructs,pharmaceutical compositions including such constructs and methods ofutilizing such constructs for down-regulating angiogenesis in tissueregions characterized by excessive or abnormal angiogenesis.

Since the present invention enables targeted expression in specific cellsubsets, it can also be modified and used in for treating varioustumors.

Thus, according to another aspect of the present invention there isprovided a method of treating tumors.

The method according to this aspect of the present invention is effectedby expressing in tumor cells the chimeric polypeptide described above.

Thus according to this aspect of the present invention, expression ofthe polypeptide chimera is directed by a tumor specific element, suchas, but not limited to, the gastrin-releasing peptide (GRP) promoter[AF293321S3; Morimoto E Anticancer Res 2001 January-February;21(1A):329-31], the hTERT promoter [AH007699; Gu J, Gene Ther 2002January; 9(1):30-7], the Hexokinase type II promoter [AF148512; Katabi MM, Hum Gene Ther. 1999 Jan. 20; 10(2):155-64.], or the L-plastinpromoter [L05490, AH002870, MMU82611; Peng X Y, Cancer Res. 2001 Jun. 1;61(11):4405-13].

Expression of the polypeptide chimera (e.g., Fas-c) in tumor cellsactivates apoptosis in these cells and thus leads to cell death, whichin turn causes tumor growth slowdown or arrest, and possibly tumorshrinkage.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures inrecombinant DNA technology described below are those well known andcommonly employed in the art. Standard techniques are used for cloning,DNA and RNA isolation, amplification and purification. Generallyenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturers'specifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989). The manual is hereinafter referred to as “Sambrook”. Othergeneral references are provided throughout this document. The procedurestherein are believed to be well known in the art and are provided forthe convenience of the reader. All the information contained therein isincorporated herein by reference.

Example 1 In-Vitro Assay for Pro-Apoptotic Gene Activity in EndothelialCells (BAEC) and 293 Cells

In cancer treatment, anti-angiogenic therapy targets the evolvingvasculature which nourishes the growing tumor [Folkman J. N Engl J Med(1995) 333(26):1757-63]. As the research of apoptosis, or programmedcell death, has progressed, numerous genes that encode selective andefficient cell death regulators have been identified [Strasser et al.Annu Rev Biochem (2000) 69:217-45.].

The present study screened several pro-apoptotic genes in order toidentify an agent most suitable for anti-angiogenic therapy. Severalpro-apoptotic genes including MORT1 (FADD—Fas associated death domainprotein, GenBank Accession number NM_(—)003824), RIP(receptor-interacting-protein, GenBank Accession number U25995), CASH(c-FLIP, GenBank Accession number AF010127), MACH (caspase 8 GenBankAccession number X98172), CPP32 (caspase 3, GenBank Accession numberU13737), caspase 9 (U60521) and Fas-chimera (Fas-c), a previouslydescribed fusion of two “death receptors”, constructed from theextracellular region of TNFR1 and the trans-membrane and intracellularregions of Fas [Boldin M P et al. J Biol Chem (1995) 270(14):7795-8, seeFIG. 1 a) were PCR amplified and cloned into the pcDNA3 (Invitrogen,Inc.) mammalian expression vector using well known prior art cloningtechniques.

These pro-apoptotic gene constructs were co-expressed along with pGFP inBAEC (Bovine Aortic Endothelial Cells) and 293 cells, which were used asnon-endothelial control cells. 24 hours post transfection, cells wereanalyzed using fluorescent microscopy. Apoptotic cells were identifiedbased on typical morphology, (i.e., small and round shape) usingfluorescence microscopy (FIGS. 2 a-b). Further assessment of theapoptotic phenotype was effected using electron microscopy (FIGS. 3a-f). Quantification of the apoptotic effect showed that MORT1, TNFR1and Fas-chimera induced the highest apoptotic activity in BAEC and 293cells (FIG. 4 a-b). Caspase 3 and 9 were less potent in this respect,probably because they were in an inactive zymogen form. Based on theseresults, the Fas-chimera (Fas-c) gene was selected for the generation ofan adenoviral-vector to be used in anti-angiogenic therapy.

Example 2 Production of Recombinant Adenoviruses Encoding Fas-ChimeraUnder the Control of the Modified PPE-1 Promoter

A cDNA encoding a full length Fas-chimera was subcloned into the plasmidpPACPPE1-3x containing the modified pre-proendothelin1 promoter (seeFIG. 1 b). Recombinant adenoviruses were produced by co-transfection ofthis plasmid with pJM17 plasmid into human embryonic kidney 293 cells.Successful viral cloning was verified via PCR amplification (FIG. 5 a).

In order to determine the expression of Fas-c in the target cells,endothelial BAEC cells were transduced with the indicated titer ofAd-PPE-Fas-c. 72 h post transduction cells were lysed and cellularproteins resolved using a non-reducing SDS-PAGE gel. Western blotanalysis was performed using anti-TNFR1 antibody (Sc-7895, Santa-CruzBiotech). As demonstrated in FIG. 5 b, a prominent band migrating at 45kD was clearly evident and its expression was dose-dependent, suggestingcorrect folding and expression of the chimeric protein. In contrast, nocorresponding bands were evident in non-transduced endothelial cells orin cells transduced with control empty viral vector. Thus, these resultsconfirmed that the adenoviral-mediated gene transfer of Fas-c results intransgene expression in the target cells.

Example 3

Ad-PPE-Fas-c Expression Induces Apoptosis in Endothelial Cells

The ability of Ad-PPE-Fas chimera to induce apoptosis of endothelialcells was determined. As shown in FIGS. 6 a-b, pre-proendothelindirected, adenovirus-mediated transduction of endothelial cells resultedin an evident and massive cell death; HUVEC and BAEC infected withAd-PPE-Fas-c (10³ MOI) had morphological features of adherent cellsundergoing apoptosis including membrane blebbing, rounding and shrinkingand detachment from the culture dish. In contrast, cells infected withcontrol viruses at the same MOI, maintained normal appearance and growthrate. Cells transduced with 100 MOI presented only a minimal degree ofcell death (data not shown).

Further assessment of the cytotoxic properties of Ad-PPE-Fas-c waseffected by expressing this virus in cells expressing the reporter geneGFP under the control of the PPE-1 promoter. As is evident from FIGS. 6c-d, most of the transduced cells acquired a typical apoptoticappearance 72 hours following transduction, whereas cells co-transducedwith control virus and Ad-PPE-GFP appeared normal.

The cytotoxic effect of Fas-c was quantified using crystal violetstaining. As shown in FIG. 7, infection of BAEC and HUVEC withAd-PPE-Fas-c resulted in mortality rates of 57% and 65%, respectively,while the control virus did not affect cell viability.

The endothelial cell specificity of the pro-apoptotic vectorAd-PPE-Fas-was demonstrated by infecting NSF (normal skin fibroblasts)with this vector. These cells, which express low levels of PPE-1[Varda-Bloom, N. et al. Gene Ther 8, 819-27. (2001)] were not affectedby infection with Ad-PPE-Fas-c. In contrast, the recombinant vectorAd-CMV-Fas-c induced apoptotic in these cells.

Example 4 Co-Administraton of Ad-PPE-Fas-c Receptor and TNFα LigandAugments the Pro-Apoptotic Effect in a Selective Manner

The ability of TNFα to augment the apoptotic effect in Fas-c expressingcells was investigated. Human TNFα was added to an endothelial cellculture 48 h-post virus infection with Ad-PPE-Fas-c (MOI of 100). Cellviability was assayed 24 h later. As shown in FIG. 8, TNFα (10 ng/ml)induced a 73% decrease in viabilty of Ad-PPE-Fas-c infected cells,whereas no significant mortality was effected by TNFα alone or in cellsinfected with control virus (Ad-Luc).

To substantiate the effect of TNFα, cell specificity was addressed. NSF(normal skin fibroblasts), DA3 (mouse mammary adenocarcinoma), D122(Lewis lung carcinoma) and B16 melanoma cells were infected withAd-PPE-Fas-c or a control virus. 48 hours later, culture wassupplemented with TNFα and cell morphology was assessed followingstaining with crystal violet. As shown in FIGS. 9 a-e, non-endothelialcells infected with Ad-PPE-Fas-c displayed normal appearance and werenot affected by TNF. On the other hand, adenoviral mediated infection ofBAEC with Fas-c resulted in marked decrease in cell viability when TNFwas added. The non-selective apoptotic activity of Fas-c driven by CMVpromoter is demonstrated in FIG. 10 a which illustrates theTNF-dependent apoptotic effect of Ad-CMV-Fas-c on endothelial cells.Viability of BAEC cells infected with the indicated MOI ofAd-CMV-Fas-chimera was determined following incubation with TNF.

Notably, the non-endothelial-specific vector Ad-CMV-Fas-c causedTNFα-dependent apoptosis of both endothelial and non-endothelial cells(FIGS. 10 b-d).

Example 5 Ad-PPE1-Fas-c Induces In-Vivo Growth Retardation of B16Melanoma in Mice

The B16 melanoma mouse model was used in order to test the anti-tumoraleffect of Fas-c expressed from the PPE1-3x promoter. B16 melanoma cells(8×10⁵) were injected subcutaneously to the flank region of 40 C57b1/6mice. When the tumor was palpable (≈5×5 mm), the mice were randomizedinto 4 groups as follows: (i) control—saline injection; (ii) controlvirus (Adeno virus containing luciferase controlled by PPE promoter);(iii) Ad-PPE1-3x-Fas-c-virus containing the Fas-TNF receptor chimericgene controlled by the preproendothelin (PPE) promoter; and (iv)Ad-CMV-Fas-c-virus containing the Fas-TNF receptor chimeric genecontrolled by the non-endothelial specific, CMV promoter.

Tumor size (length and width) was measured using a hand caliper. Asshown in FIG. 11 a, tumor size was lower for mice treated withAd-PPE1-3x-Fas-c or Ad-CMV-Fas-c as compared to control mice. Tumorweights at the end of the treatment period was also lower in theAd-PPE1-3x-Fas-c treated mice (FIG. 11 b). Mice injected withAd-PPE1-3x-Fas-c showed marked necrosis of their tumor (FIG. 11 c).

Example 6 Modifications of the PPE Promoter

The modified murine PPE-1 promoter was developed by inserting threecopies of the positive transcription element discovered by Bu et al (J.Biol Chem. (1997) 272(19): 32613-32622) into the Nhel restriction enzymesite located downstream (−286 bp) to the 43 base pairs endogenouspositive element (−364 to −320 bp).

The enhancer fragment termed herein “3X” is a triplicate copy of anendogenous sequence element (nucleotide coordinates 407-452 of SEQ IDNO:1) present in the murine PPE-1 promoter. It has been previously shownthat induction of PPE-1 promoter activity in vascular endothelial cellsdepends on the presence of this element Bu et al (J. Biol Chem. (1997)272(19): 32613-32622). The 3X fragment was synthesized by using twocomplementary single stranded DNA strands 96 base pares in length(BioTechnology industries; Nes Tziona, Israel), (SEQ ID NO: 2 and 3).The two single stranded DNA fragment were annealed and filled usingKlenow fragment (NEB); the resulting double stranded DNA was 145 basepairs long and included Nhe-1 restriction sites (SEQ ID NO: 4).

The 3X fragment was ligated into the murine PPE-1 promoter down streamof endogenous Nhe-1 site using T4 Ligase. The resulting construct waspropagated in DH5α compatent cells and a large-scale plasmid preparationwas produced using the maxi-prep Qiagene kit.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

1-26. (canceled)
 27. A vector comprising a nucleic acid construct, whichcomprises: a) a first polynucleotide region encoding a chimericpolypeptide comprising a ligand binding domain of TNF Receptor 1 (TNFR1)fused to an effector domain of Fas; and b) a second polynucleotideregion comprising a PPE-1-3X promoter, which directs expression of thechimeric polypeptide in endothelial cells of angiogenic blood vessels;wherein the chimeric polypeptide, when expressed, induces apoptosis ofthe endothelial cells.
 28. The vector of claim 27, wherein the ligandbinding domain of TNFR1 comprises an extracellular domain of TNFR1. 29.The vector of claim 27, wherein the effector domain of Fas comprises atrans-membrane region and an intracellular region of Fas.
 30. The vectorof claim 27, wherein the ligand binding domain of TNFR1 is anextracellular domain of TNFR1 and the effector domain of Fas is atrans-membrane region and an intracellular region of Fas.
 31. The vectorof claim 30, wherein the vector is an adenoviral vector.
 32. The vectorof claim 31, wherein the adenoviral vector is adenovirus serotype-5. 33.The vector of claim 31, wherein the vector comprisesAd-PPE-1-3X-Fas-chimera.
 34. The vector of claim 27, wherein theapoptosis of the endothelial cells results in a down-regulation ofangiogenesis in a tumor.
 35. The vector of claim 27, wherein theapoptosis of the endothelial cells results in a reduction in the size ofa tumor.
 36. A pharmaceutical composition comprising the vector of claim27 and a pharmaceutically acceptable carrier.
 37. A method of producinga vector comprising transducing a host cell with a vector comprising anucleic acid construct which comprises: a) a first polynucleotide regionencoding a chimeric polypeptide comprising a ligand binding domain ofTNF Receptor 1 (TNFR1) fused to an effector domain of Fas; and b) asecond polynucleotide region comprising a PPE-1-3X promoter, whichdirects expression of the chimeric polypeptide in endothelial cells ofangiogenic blood vessels; wherein the chimeric polypeptide, whenexpressed, induces apoptosis of the endothelial cells.
 38. The method ofclaim 37, wherein the ligand binding domain of TNFR1 comprises anextracellular domain of TNFR1.
 39. The method of claim 37, wherein theeffector domain of Fas comprises a trans-membrane region and anintracellular region of Fas.
 40. The method of claim 37, wherein theligand binding domain of TNFR1 is an extracellular domain of TNFR1 andthe effector domain of Fas is a trans-membrane region and anintracellular region of Fas.
 41. The method of claim 37, wherein thevector is an adenoviral vector.
 42. The method of claim 41, wherein theadenoviral vector is adenovirus serotype-5.
 43. The method of claim 41,wherein the vector comprises Ad-PPE-1-3X-Fas-chimera.
 44. A method oftreating a tumor in a subject in need thereof, the method comprisingadministering to the subject a vector comprising a nucleic acidconstruct comprising: a) a first polynucleotide region encoding achimeric polypeptide comprising a ligand binding domain of TNF Receptor1 (TNFR1) fused to an effector domain of Fas; and b) a secondpolynucleotide region comprising a PPE-1-3X promoter, which directsexpression of the chimeric polypeptide in endothelial cells ofangiogenic blood vessels in a tumor; wherein the chimeric polypeptide,when expressed, induces apoptosis of the endothelial cells in the tumorof the subject and wherein the vector is administered systemically orlocally.
 45. The method of claim 44, further comprising administeringTNFα to the subject.
 46. The method of claim 44, wherein theadministering is parenteral administration.